Antimicrobial resistance and plasmid detection in strains of the Bacteroides fragilis group

Resistant populations of the Bacteroides fragilis group bacteria (two reference ones and two isolated from human and Callithrix penicillata marmoset) were obtained by the gradient plate technique, to clindamycin, penicillin G, metronidazole and mercuric chloride. All the four tested strains were originaly susceptible to the four antimicrobial drugs at the breakpoint used in this study. MICs determination for the four cultures gave constant values for each antimicrobial, on the several steps by the gradient plate technique. The intestinal human B. fragilis strains showed three DNA bands, that could be representative of only two plasmids in the closed covalently circular (CCC) form with molecular weights of approximately 25 and 2.5 Md. The results do not permit an association between the presence of plasmid in the human strain with the susceptibility to the studied drugs. The four strains were ß-lactamase negative in the two methods used, and no particular chromosomal genetic resistance marker was demonstred. The resistance (MIC) observed, after contact with penicillin G and mercuric chloride, were two-fold in the four tested strains

Liaisons dangereuses, conservation of modern and contemporary art: a study of the synthetic binding media in Portugal

Dissertação apresentada para obtenção do Grau de Doutor em Conservação e Restauro, especialidade de Ciências da Conservação, pela Universidade Nova de Lisboa, Faculdade de Ciências e Tecnologia

Serological follow-up of patients with paracoccidioidomycosis treated with itraconazole using Dot-blot, ELISA and Western-blot

Twenty-seven mycologically proven cases of paracoccidioidomycosis (PCM) were treated with itraconazole (100-200 mg/day in month 1 and 100 mg/day until month 6-8) and evaluated clinically and serologically, up to 3.5 years post-therapy, using Dot-blot and ELISA for measuring the titers of IgG, IgA and IgM anti-P. brasiliensis antibodies and Western-blot for determining IgG, IgA and IgM antibodies against the antigen components of the fungus. Before treatment, 81.5% (Dot-blot) and 84% (ELISA) of the patients presented elevated IgG anti-P. brasiliensis antibody titers which dropped slightly with treatment. On the other hand, the percentages of pre-treatment high-titered sera for IgA and IgM anti-P.brasiliensis were lower (5l.9% and 5l.8%: Dot-blot; 16.5 and 36%: ELISA, respectively) but the titers tended to become negative more frequently with treatment. Prior to treatment, the percentages of positivity for IgG, IgA and IgM anti-P.brasiliensis antibodies in Western-blot were 96%, 20.8% and 41.6%, respectively. Antigens with molecular weights varying from 16-78 kDa, from 21-76 kDa and from 27-78 kDa were reactive for IgG, IgA and IgM antibodies, respectively. The most frequently reactive antigenic components had molecular weights of 27, 33 and 43 kDa for IgG, and 70 for IgA and IgM antibodies. During the period of study, the patients responded well to treatment. The present data confirm the diversity and complexity of the humoral response in PCM, and the importance of utilizing different serological tests to detect IgG, IgA and IgM anti-P. brasiliensis antibodies

Excretion-secretion products and proteases from live Sporothrix schenckii yeast phase: immunological detection and cleavage of human IgG

Antigenic preparations from Sporothrix schenckii usually involve materials from mixed cultures of yeast and mycelia presenting cross-reactions with other deep mycoses. We have standardized pure yeast phase with high viability of the cells suitable to obtain specific excretion-secretion products without somatic contaminations. These excretion-secretion products were highly immunogenic and did not produce noticeable cross-reactions in either double immunodiffusion or Western blot. The antigenic preparation consists mainly of proteins with molecular weights between 40 and 70 kDa, some of them with proteolytic activity in mild acidic conditions. We also observed cathepsin-like activity at two days of culture and chymotrypsin-like activity at four days of culture consistent with the change in concentration of different secreted proteins. The proteases were able to cleave different subclasses of human IgG suggesting a sequential production of antigens and molecules that could interact and interfere with the immune response of the host.

Selective synthesis under microwave irradiation of new monomers for potential applications in PDLC films

Dissertation to obtain the degree of master in Bioorganic

Identification and purification of immunogenic proteins from nonliving promastigote polyvalent Leishmania vaccine (Leishvacin®)

Immunogenic proteins from nonliving promastigote polyvalent Leishmania vaccine against American tegumentary leishmaniasis (Leishvacin®), produced by Biobrás (Biochemistry of Brazil ), Montes Claros, State of Minas Gerais, Brazil, were identified and purified by polyacrylamide electrophoresis gel and electroelution. C57BL/10 mice were vaccinated with proteins with estimated molecular weights of 42, 46, 63, 66, 73, 87, 97, and 160kDa in three doses of 30µg of each protein at 15-day intervals combined with 250µg of Corynebacterium parvum followed by a challenge infection with 10(5) infective promastigotes from Leishmania (Leishmania) amazonensis. The ability of these proteins to induce immune response and protection was analyzed. No statistical difference was observed in the level of IFN-g induced by proteins in vaccinated groups in comparison with control groups. Six months after challenge infection, protection levels of 28.57; 42.86; 57.14; 42.86; 42.86, 57.14; 42.86 and 57.14% were demonstrated for each purified protein.

Detection of immunogenic proteins from Anopheles sundaicussalivary glands in the human serum

AbstractINTRODUCTION:The saliva of mosquitoes has an important role in the transmission of several diseases, including malaria, and contains substances with vasomodulating and immunomodulating effects to counteract the host physiological mechanisms and enhance pathogen transmission. As immunomodulatory components, salivary gland proteins can induce the generation of specific IgG antibodies in the host, which can be used as specific biomarkers of exposure to Anopheles sundaicus . The objective of this study was to identify immunogenic proteins from the salivary glands of Anopheles sundaicus by reaction with sera from individuals living in malaria-endemic areas who are thus exposed to Anopheles mosquitoes.METHODS:IgG antibodies targeting salivary gland proteins in serum samples from individuals living in malaria-endemic areas were measured by enzyme-linked immunosorbent assay (ELISA). Sera from healthy individuals living in non-endemic areas were used as negative controls. Determination of the presence of salivary gland immunogenic proteins was carried out by western blotting.RESULTS:Sixteen bands appeared in sodium dodecyl sulfate polyacrylamide gel electrophoresis, with molecule weights ranging from 22 to 144kDa. Among the exposed individuals, IgG responses to salivary gland proteins were variable. Protein bands with molecular weights of 46, 41, 33, and 31kDa were the most immunogenic. These immunogenic proteins were consistently recognized by pooled serum and individual samples from people living in malaria-endemic areas but not by negative controls.CONCLUSIONS:These results support the potential use of immunogenic proteins from the salivary glands of Anopheles as candidate markers of bite exposure or in malaria vaccines.

PEGylated DOTA-AHA-based Gd(III) chelates – A relaxometric study

Three PEGylated derivatives of 1,4,7,10-tetraazacyclododecane-1-((6-amino)hexanoic)-4,7,10-triacetic acid) (DOTA-AHA) with different molecular weights were prepared and characterized. Their Gd(III) chelates were studied in aqueous solution using variable-temperature 1H nuclear magnetic relaxation dispersion (NMRD) and 17ONMR spectroscopy in view of the determination of their relaxivity and the parameters that govern it. The relaxivity varied from 5.1 to 6.5 mM-1.s-1 (37 ºC and 60 MHz) with the increasing molecular weight of the PEG chain, being slightly higher than that of the parent chelate Gd(DOTA-AHA), due to a small contribution of a slow global rotation of the complexes. A variable temperature 1H NMR study of several Ln(III) chelates of DOTA-A(PEG750)HA allowed the determination of the isomeric M/m ratio (M = square antiprismatic isomer and m = twisted square antiprismatic isomer, the latter presenting a much faster water exchange) which for the Gd(III) chelate was estimated in circa 1:0.2, very close to that of [Gd(DOTA)]-. This explains why the PEGylated Gd(III) chelate has a water rate exchange similar to that of [Gd(DOTA)]-. The predominance of the M isomer is a consequence of the bulky PEG moiety which does not favor the stabilization of the m isomer in sterically crowded systems at the substituent site, contrary to what happens with less packed asymmetrical DOTA-type chelates with substitution in one of the four acetate C(α) atoms.

Nanocomposites: the contribution of multiscale modelling to improve the dispersion levels

Tese de Doutoramento em Ciência e Engenharia de Polímeros e Compósitos

Trypanosoma cruzi: identification of specific epimastigote antigens by human immune sera

Soluble antigens from epimastigotes of Trypanosoma cruzi were analyzed by western blot in terms of their reactivity with sera from patients with Chagas' disease. In addition, sera from patients with visceral (AVL) and tegumentar leishmaniasis (ATL) were also tested in order to identify cross-reactivities with Trypanosoma cruzy antigens. Twenty eight polypeptides with molecular weights ranging from 14 kDa to 113 kDa were identified with sera from Chagas' disease patients. An extensive cross-reactivity was observed when sera from human visceral leishmaniasis were used, while only a slight cross-reaction was observed with sera from tegumentar leishmaniasis. On the other hand, 10 polypeptidesspecifically reacting with sera from Chagas' disease patients were identified. Among them, the antigens with molecular weights of 46 kDa and 25 kDa reacted with all sera teste and may be good candidates for specific immunodiagnosis of Chagas' disease.

Strain specific variation of outer membrane proteins of wild Yersinia pestis strains subjected to different growth temperatures

Three Yersinia pestis strains isolated from humans and one laboratory strain (EV76) were grown in rich media at 28§C and 37§C and their outer membrane protein composition compared by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-Page). Several proteins with molecular weights ranging from 34 kDa to 7 kDa were observed to change in relative abundance in samples grown at different temperatures. At least seven Y. pestis outer membrane proteins showed a temperature-dependent and strain-specific behaviour. Some differences between the outer membrane proteins of full-pathogenic wild isolates and the EV76 strain could aldso be detected and the relevance of this finding on the use of laboratory strains as a reference to the study of Y. pestis biological properties is discuted.

The p53 gene expression and its developmental regulation in schistosomes

We have studied the gene expression, especially of the oncoproteins, and its regulation in schistosomes. Schistosomes have a complex life cycle with defined dimorphic lifestyle. The parasite are so far unique in biology in expressing oncogene products in their adult stage. In order to characterize the expression and developmental regulation, a lambda gt 11 cDNA library and lambda EMBL4 genomic DNA library of each growth stage of Schistosoma mansoni and S. japonicum was constructed, and was screened with various monoclonal antibodies against ongogene products. One positive plaque reacted to anti-p53 antibody (Ab-2, Oncogene Science, Inc.) was further analyzed. This fusion protein was about 120 KDa in molecular weights, and expressed as 1.4 Kb RNA in the adult stage. P53 gene is well-known as the negative regulator of the cell cicle, and the mutations in the gene are turning out to be the most common genetic alterations in human cancers. The comparison of the gene structure among species and stages were being conducted. Chromosome structures, C-band formation, and the results of in situ hybridization using the phage probe would be discussed.

Mayaro virus proteins

Mayaro virus was grown in BHK-21 cells and purified by centrifugation in a potassium-tartrate gradient (5-50%). The electron microscopy analyses of the purified virus showed an homogeneous population of enveloped particles with 69 ñ 2.3 nm in diameter. Three structural virus proteins were identified and designated pl, p2 and p3. Their average molecular weight were p1, 54 KDa; p2, 50 KDa and p3, 34 KDa. In Mayaro virus infected. Aedes albopictus cells and in BHK-21 infected cells we detected six viral proteins, in wich three of them are the structural virus proteins and the other three were products from processing of precursors of viral proteins, whose molecular weights are 62 KDa, 64 KDa and 110 KDa. The 34 KDa protein was the first viral protein sinthesized at 5 hours post-infection in both cell lines studied.

Differential distribution of tau proteins in developing cat cerebellum.

The differential distribution and phosphorylation of tau proteins in cat cerebellum was studied with two well characterized antibodies, TAU-1 and TAU-2. TAU-1 detects tau proteins in axons, and the epitope in perikarya and dendrites is masked by phosphorylation. TAU-2 detects a phosphorylation-independent epitope on tau proteins. The molecular composition of tau proteins in the range of 45 kD to 64 kD at birth changed after the first postnatal month to a set of several adult variants of higher molecular weights in the range of 59 kD to 95 kD. The appearance of tau proteins in subsets of axons corresponds to the axonal maturation of cerebellar local-circuit neurons in granular and molecular layers and confirms previous studies. Tau proteins were also identified in synapses by immunofluorescent double-staining with synapsin I, located in the pinceau around the Purkinje cells, and in glomeruli. Dephosphorylation of juvenile cerebellar tissue by alkaline phosphatase indicated indirectly the presence of differentially phosphorylated tau forms mainly in juvenile ages. Additional TAU-1 immunoreactivity was unmasked in numerous perikarya and dendrites of stellate cells, and in cell bodies of granule cells. Purkinje cell bodies were stained transiently at juvenile ages. During postnatal development, the intensity of the phosphate-dependent staining decreased, suggesting that phosphorylation of tau proteins in perikarya and dendrites may be essential for early steps in neuronal morphogenesis during cat cerebellum development.

Haemolymph and Fat Body Metallo-protease Associated with Enterobacter cloacae Infection in the Bloodsucking Insect, Rhodnius prolixus

Analysis of zymograms with SDS-polyacrilamide gel electrophoresis containing gelatin as substrate, and performed on samples of haemolymph or fat body taken from Rhodnius prolixus inoculated or not with Enterobacter cloacae, demonstrated distinct patterns of protease activities: (i) in the haemolymph two proteases were induced in insects inoculated with bacteria; (ii) two proteases were detected in the fat bodies derived from non-inoculated controls or insect inoculated with sterile culture medium; (iii) haemolymph and fat body had both the same apparent molecular weights proteases (46 and 56 kDa); and (iv) these enzymes were characterized as metallo-proteases. The association of these enzymes in Rhodnius infected with bacteria was discussed.